CADASIL-CAUSING NOTCH3 MUTATION CONTRIBUTES TO THE DEVELOPMENT OF PULMONARY ARTERIAL HYPERTENSION IN MALES

Abstract

Objective: Notch3 signalling has been associated with changes in the pulmonary vasculature leading to pulmonary arterial hypertension (PAH). Mice with the CADASIL-causing R169C Notch3 mutation (TgNotch3R169C) exhibit gain-of-function Notch3 signalling and develop peripheral vascular dysfunction and injury. Here we investigated whether the R169C mutation contributes to PAH development. Design and method: Briefly, 24-week male and female mice overexpressing either a WT or R169C mutant Notch3 gene were exposed to 2 weeks normoxia (N) or chronic hypobaric hypoxia (10% O2) (H). PAH was assessed by in vivo haemodynamic measurements, Fulton index analysis of right ventricle hypertrophy (RVH), and histological remodelling analysis by elastin staining of pulmonary arteries under 80 microns in diameter. Pulmonary artery wire myography was also performed. Results: RV systolic pressure increased in hypoxic animals vs normoxic (WT H 27.5 ± 1.0 vs WT N 22.8 ± 2.2mmHg); and was exacerbated only in male R169C hypoxic animals (R169C H 32.2 ± 1.1 mmHg vs R169C N 23.6 ± 1.4) (p < 0.05). Male R169C hypoxic animals also exhibited significant RV hypertrophy (H 0.26 ± 0.033 vs N 0.16 ± 0.012), p < 0.05. RVH was not significantly different between female groups. R169C pulmonary arteries from males were significantly hypercontractile to 5HT (Emax: R169C 142 ± 10% vs WT 92 ± 6%); an effect attenuated by ROCK inhibitor Fasudil (Emax: R169C ± Fas. 117 ± 20%). Female R169C arteries were not hypercontractile to 5HT (R169C 113.3 ± 11% vs WT 120.3 ± 8%) but Fasudil reduced contraction (Emax: R169C + Fas. 96.8 ± 10%). Notch3 effector gene Hes5, implicated in PAH, was significantly increased in male R169C lung (8.7 fold change vs WT) suggesting increased Notch3 activation. RhoA/ROCK activating Rho-guanine nucleotide exchange factors LARG and PDZ mRNA were upregulated in R169C lung (R169C 33.5 fold vs WT, R169C 18.7 fold vs WT respectively). These changes were not observed in lungs from female mice. Conclusions: Notch3 R169C mutation may contribute to PAH susceptibility and pulmonary vascular hypercontractility in males. ROCK signaling plays an important role in these processes.